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以番茄叶片为试材,采用改进的CTAB法,电钻研磨,提取过程中加入醋酸铜,设计4因素3水平的正交试验对PCR反应体系进行优化,同时利用梯度PCR对退火温度进行选择选择。结果表明:优化的10μL反应体系含:1×buffer,1.2 mmol/L Mg2+,1.5 mmol/L dNTPs,1.2μmol/L引物,0.75 UTaqDNA聚合酶,10 ng模板DNA。扩增程序为:94℃预变性2 min;94℃变性30 s,49.2℃退火30 s,68℃延伸30 s,共35个循环;最后72℃延伸8 min。优化的反应体系可以用于SSR分子标记机的研究,在所有SSR引物中基本都能有效扩增;改进的CTAB法提取的DNA纯度更高。
The tomato leaves were used as experimental material. The modified CTAB method and electric drill were used for grinding. During the extraction process, copper acetate was added. The orthogonal experiment of 4 factors and 3 levels was designed to optimize the PCR reaction system. At the same time, the selection of annealing temperature was carried out by gradient PCR. The results showed that the optimal reaction system consisted of 1 × buffer, 1.2 mmol / L Mg2 +, 1.5 mmol / L dNTPs, 1.2 μmol / L primer, 0.75 UTaq DNA polymerase and 10 ng template DNA. The amplification program was: denaturation at 94 ° C for 2 min; denaturation at 94 ° C for 30 s, annealing at 49.2 ° C for 30 s, extension at 68 ° C for 30 s for a total of 35 cycles; and final extension at 72 ° C for 8 min. The optimized reaction system can be used in the research of SSR molecular marker, and can be effectively amplified in all SSR primers. The DNA extracted by the improved CTAB method is more pure.